Heterocyclic substituted benzothiophenes, compositions, and methods

ABSTRACT

The present invention provides class of compounds of formula I ##STR1## and their pharmaceutically acceptable salts in which R 1  is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkylcarbonyloxy, arylcarbonyloxy; R 2  is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkylcarbonyloxy, alkylsulfonyloxy, arylcarbonyloxy, Cl or Br; R 3  and R 4  are independently C 1  -C 4  alkyl, or combine with the nitrogen atom to which they are attached to form a pyrrolidino, piperidino, or hexamethyleneimino ring; X is selected from ##STR2## and Y is --CO--, --CHOH--, or --CH 2  --. The compounds and pharmaceutical compositions containing the compounds, either alone or in combination with either progestin or estrogen, are useful for alleviating the symptoms of osteoporosis, cardiovascular related pathological conditions such as hyperlipidemia, and estrogen-dependent cancer. 
     The compounds of the present invention also are useful for inhibiting uterine fibroid disease and endometriosis in women and aortal smooth muscle cell proliferation, particularly restenosis, in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Provisional Application SerialNo. 60/013,291 filed Mar. 12, 1996.

FIELD OF THE INVENTION

The present invention relates to organic compounds having biologicalactivity, to compositions comprising the compounds, and to medicalmethods of treatment. More particularly, the present invention concernsa class of heterocyclic substituted benzothiophene compounds, topharmaceutical compositions comprising the compounds, and to methods oftreating conditions associated with post-menopausal syndrome, includingosteoporosis, cardiovascular disease (particularly hyperlipidemia),hormonally dependent cancers (particularly of the breast and uterine)and conditions not necessarily associated with post-menopause includinguterine fibroid disease, endometriosis, and aortal smooth muscle cellproliferation.

BACKGROUND OF THE INVENTION

"Post-menopausal syndrome" is a term used to describe variouspathological conditions which frequently affect women who have enteredinto or completed the physiological metamorphosis known as menopause.Although numerous pathologies are contemplated by the use of this term,three major effects of post-menopausal syndrome are the source of thegreatest long-term medical concern: osteoporosis, cardiovascular effectssuch as hyperlipidemia, and estrogen-dependent cancer, particularlybreast and uterine cancer.

Osteoporosis describes a group of diseases which arise from diverseetiologies, but which are characterized by the net loss of bone mass perunit volume. The consequence of this loss of bone mass and resultingbone fracture is the failure of the skeleton to provide adequatestructural support for the body. One of the most common types ofosteoporosis is that associated with menopause. Most women lose fromabout 20% to about 60% of the bone mass in the trabecular compartment ofthe bone within 3 to 6 years after the cessation of menses. This rapidloss is generally associated with an increase of bone resorption andformation. However, the resorptive cycle is more dominant and the resultis a net loss of bone mass. Osteoporosis is a common and serious diseaseamong post-menopausal women.

There are an estimated 25 million women in the United States, alone, whoare afflicted with this disease. The results of osteoporosis arepersonally harmful and also account for a large economic loss due itschronicity and the need for extensive and long term support(hospitalization and nursing home care) from the disease sequelae. Thisis especially true in more elderly patients. Additionally, althoughosteoporosis is not generally thought of as a life threateningcondition, a 20% to 30% mortality rate is related with hip fractures inelderly women. A large percentage of this mortality rate can be directlyassociated with post-menopausal osteoporosis.

The most vulnerable tissue in the bone to the effects of post-menopausalosteoporosis is the trabecular bone. This tissue is often referred to asspongy or cancellous bone and is particularly concentrated near the endsof the bone (near the joints) and in the vertebrae of the spine. Thetrabecular tissue is characterized by small osteoid structures whichinter-connect with each other, as well as the more solid and densecortical tissue which makes up the outer surface and central shaft ofthe bone. This inter-connected network of trabeculae gives lateralsupport to the outer cortical structure and is critical to thebio-mechanical strength of the overall structure. In post-menopausalosteoporosis, it is, primarily, the net resorption and loss of thetrabeculae which leads to the failure and fracture of bone. In light ofthe loss of the trabeculae in post-menopausal women, it is notsurprising that the most common fractures are those associated withbones which are highly dependent on trabecular support, e.g., thevertebrae, the neck of the weight bearing bones such as the femur andthe fore-arm. Indeed, hip fracture, collies fractures, and vertebralcrush fractures are hall-marks of post-menopausal osteoporosis.

At this time, the generally accepted method for treatment ofpost-menopausal osteoporosis is estrogen replacement therapy. Althoughtherapy is generally successful, patient compliance with the therapy islow primarily because estrogen treatment frequently produces undesirableside effects.

Throughout premenopausal time, most women have less incidence ofcardiovascular disease than age-matched men. Following menopause,however, the rate of cardiovascular disease in women slowly increases tomatch the rate seen in men. This loss of protection has been linked tothe loss of estrogen and, in particular, to the loss of estrogen'sability to regulate the levels of serum lipids. The nature of estrogen'sability to regulate serum lipids is not well understood, but evidence todate indicates that estrogen can upregulate the low density lipid (LDL)receptors in the liver to remove excess cholesterol. Additionally,estrogen appears to have some effect on the biosynthesis of cholesterol,and other beneficial effects on cardiovascular health.

It has been reported in the literature that post-menopausal women havingestrogen replacement therapy have a return of serum lipid levels toconcentrations to those of the pre-menopausal state. Thus, estrogenwould appear to be a reasonable treatment for this condition. However,the side-effects of estrogen replacement therapy are not acceptable tomany women, thus limiting the use of this therapy. An ideal therapy forthis condition would be an agent which would regulate the serum lipidlevel as does estrogen, but would be devoid of the side-effects andrisks associated with estrogen therapy.

The third major pathology associated with post-menopausal syndrome isestrogen-dependent breast cancer and, to a lesser extent,estrogen-dependent cancers of other organs, particularly the uterus.Although such neoplasms are not solely limited to a post-menopausalwomen, they are more prevalent in the older, post-menopausal population.Current chemotherapy of these cancers has relied heavily on the use ofanti-estrogen compounds such as, for example, tamoxifen. Although suchmixed agonist-antagonists have beneficial effects in the treatment ofthese cancers, and the estrogenic side-effects are tolerable in acutelife-threatening situations, they are not ideal. For example, theseagents may have stimulatory effects on certain cancer cell populationsin the uterus due to their estrogenic (agonist) properties and they may,therefore, be contraproductive in some cases. A better therapy for thetreatment of these cancers would be an agent which is an anti-estrogencompound having negligible or no estrogen agonist properties onreproductive tissues.

In response to the clear need for new pharmaceutical agents which arecapable of alleviating the symptoms of, inter alia, post-menopausalsyndrome, the present invention provides benzothiophene compounds,pharmaceutical compositions thereof, and methods of using such compoundsfor the treatment of post-menopausal syndrome and other estrogen-relatedpathological conditions such as those mentioned below.

Uterine fibrosis (uterine fibroid disease) is an old and ever presentclinical problem which goes under a variety of names, including uterinefibroid disease, uterine hypertrophy, uterine lieomyomata, myometrialhypertrophy, fibrosis uteri, and fibrotic metritis. Essentially, uterinefibrosis is a condition where there is an inappropriate deposition offibroid tissue on the wall of the uterus.

This condition is a cause of dysmenorrhea and infertility in women. Theexact cause of this condition is poorly understood but evidence suggeststhat it is an inappropriate response of fibroid tissue to estrogen. Sucha condition has been produced in rabbits by daily administrations ofestrogen for 3 months. In guinea pigs, the condition has been producedby daily administration of estrogen for four months. Further, in rats,estrogen causes similar hypertrophy.

The most common treatment of uterine fibrosis involves surgicalprocedures both costly and sometimes a source of complications such asthe formation of abdominal adhesions and infections. In some patients,initial surgery is only a temporary treatment and the fibroids regrow.In those cases a hysterectomy is performed which effectively ends thefibroids but also the reproductive life of the patient. Also,gonadotropin releasing hormone antagonists may be administered, yettheir use is tempered by the fact they can lead to osteoporosis. Thus,there exists a need for new methods for treating uterine fibrosis, andthe methods of the present invention satisfy that need.

Endometriosis is a condition of severe dysmenorrhea, which isaccompanied by severe pain, bleeding into the endometrial masses orperitoneal cavity and often leads to infertility. The cause of thesymptoms of this condition appear to be ectopic endometrial growthswhich respond inappropriately to normal hormonal control and are locatedin inappropriate tissues. Because of the inappropriate locations forendometrial growth, the tissue seems to initiate local inflammatory-likeresponses causing macrophage infiltration and a cascade of eventsleading to initiation of the painful response. The exact etiology ofthis disease is not well understood and its treatment by hormonaltherapy is diverse, poorly defined, and marked by numerous unwanted andperhaps dangerous side effects.

One of the treatments for this disease is the use of low dose estrogento suppress endometrial growth through a negative feedback effect oncentral gonadotropin release and subsequent ovarian production ofestrogen; however, it is sometimes necessary to use continuous estrogento control the symptoms. This use of estrogen can often lead toundesirable side effects and even the risk of endometrial cancer.

Another treatment consists of continuous administration of progestinswhich induces amenorrhea and by suppressing ovarian estrogen productioncan cause regressions of the endometrial growths. The use of chronicprogestin therapy is often accompanied by the unpleasant CNS sideeffects of progestins and often leads to infertility due to suppressionof ovarian function.

A third treatment consists of the administration of weak androgens,which are effective in controlling the endometriosis; however, theyinduce severe masculinizing effects. Several of these treatments forendometriosis have also been implicated in causing a mild degree of boneloss with continued therapy. Therefore, new methods of treatingendometriosis are desirable.

Smooth aortal muscle cell proliferation plays an important role indiseases such as atherosclerosis and restenosis. Vascular restenosisafter percutaneous transluminal coronary angioplasty (PTCA) has beenshown to be a tissue response characterized by an early and late phase.The early phase occurring hours to days after PTCA is due to thrombosiswith some vasospasms while the late phase appears to be dominated byexcessive proliferation and migration of aortal smooth muscle cells. Inthis disease, the increased cell motility and colonization by suchmuscle cells and macrophages contribute significantly to thepathogenesis of the disease. The excessive proliferation and migrationof vascular aortal smooth muscle cells may be the primary mechanism tothe reocclusion of coronary arteries following PTCA, atherectomy, laserangioplasty and arterial bypass graft surgery. See "IntimalProliferation of Smooth Muscle Cells as an Explanation for RecurrentCoronary Artery Stenosis after Percutaneous Transluminal CoronaryAngioplasty," Austin et al., Journal of the American College ofCardiology, 8:369-375 (August 1985).

Vascular restenosis remains a major long term complication followingsurgical intervention of blocked arteries by percutaneous transluminalcoronary angioplasty (PTCA), atherectomy, laser angioplasty and arterialbypass graft surgery. In about 35% of the patients who undergo PTCA,reocclusion occurs within three to six months after the procedure. Thecurrent strategies for treating vascular restenosis include mechanicalintervention by devices such as stents or pharmacologic therapiesincluding heparin, low molecular weight heparin, coumarin, aspirin, fishoil, calcium antagonist, steroids, and prostacyclin. These strategieshave failed to curb the reocclusion rate and have been ineffective forthe treatment and prevention of vascular restenosis. See "Prevention ofRestenosis after Percutaneous Transluminal Coronary Angioplasty: TheSearch for a `Magic Bullet`,"Hermans et al., American Heart Journal,122:171-187 (July 1991).

In the pathogenesis of restenosis excessive cell proliferation andmigration occurs as a result of growth factors produced by cellularconstituents in the blood and the damaged arterial vessel wall whichmediate the proliferation of smooth muscle cells in vascular restenosis.

Agents that inhibit the proliferation and/or migration of smooth aortalmuscle cells are useful in the treatment and prevention of restenosis.The present invention provides for the use of compounds as smooth aortalmuscle cell proliferation inhibitors and, thus inhibitors of restenosis.

SUMMARY OF THE INVENTION

In its principal embodiment, the present invention provides a class ofsubstituted benzo b!thiophene compounds of formula I ##STR3## or apharmaceutically acceptable salt thereof in which R¹ is selected fromthe group consisting of hydrogen, hydroxy, C₁ -C₄ alkoxy, ##STR4## (inwhich Ar is optionally substituted phenyl), and --OSO₂ (C₄ -C₆ straightchain alkyl).

R² is selected from the group consisting of hydrogen, hydroxy, C₁ -C₄alkoxy, ##STR5## ##STR6## Cl and Br.

The substituent groups R³ and R⁴ are independently C₁ -C₄ alkyl, orcombine to form, together with the nitrogen atom to which they areattached, a piperidino, pyrrolidino, or hexamethyleneimino ring;

X is selected from the group consisting of ##STR7##

Y is selected from the group consisting of --CO--, --CHOH--, or --CH₂--.

The present invention further relates to pharmaceutical compositionscontaining compounds of formula I, optionally containing estrogen orprogestin, and the use of such compounds, alone, or in combination withestrogen or progestin, for alleviating the symptoms of post-menopausalsyndrome, particularly osteoporosis, cardiovascular related pathologicalconditions, and estrogen-dependent cancer.

The compounds of the present invention also are useful for inhibitinguterine fibroid disease and endometriosis in women and aortal smoothmuscle cell proliferation in humans.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been found that aselect group of 2-aryl-3-heterocyclic benzo b!thiophenes, i.e., thecompounds of formula I, are useful for the treatment or prevention ofthe symptoms and pathologies of: post-menopausal syndrome: osteoporosis,hyperlipidemia, estrogen dependent cancers, uterine fibroids,endometriosis, or restenosis in mammals, including humans.

As used throughout this specification and the appended claims, the term"C₁ -C₆ alkyl" represents a straight or branched alkyl chain having from1 to 6 carbon atoms. Typical C₁ -C₆ alkyl groups include methyl, ethyl,n-propyl, n-hexyl, and n-butyl. The term "C₁ -C₄ alkoxy" representsgroups such as methoxy, ethoxy, n-propoxy, and n-butoxy.

"Optionally substituted phenyl" is meant to denote unsubstituted phenyland phenyl substituted with one or two substituent groupos independentlyselected from C₁ -C₆ alkyl, C₁ -C₄ alkoxy, hydroxy, nitro, chloro,fluoro, and trichloro- or trifluoromethyl.

The term "solvate" represents an aggregate that comprises one or moremolecules of the solute, such as a formula I compound, with a moleculeof solvent.

The term "inhibit" is defined to include its generally accepted meaningwhich includes prohibiting preventing, restraining, and slowing,stopping or reversing progression, or severity, or such action on aresultant symptom. As such, the present invention includes both medicaltherapeutic and/or prophylactic administration, as appropriate.

As used herein, the term "estrogen" includes steroidal compounds havingestrogenic activity such as, for example, 17β-estradiol, estrone,conjugated estrogen (Premarin®), equine estrogen, 17-αethynyl estradiol,and the like. As used herein, the term "progestin" includes compoundshaving progestational activity such as, for example, progesterone,norethylnodrel, nongestrel, megestrol acetate, norethindrone, and thelike.

While the scope of the chemical compound aspect of the present inventionis defined by formula I above, specific examples of compounds fallingwithin the invention include, but are not necessarily limited to:

2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 2-2-(1-piperidinyl)ethoxy!pyrimidin-5-yl!methanone,

2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 5-2-(1-piperidinyl)pyrimidin-2-yl!methanone,

2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 2-2-(1-piperidinyl)ethoxy!pyrazin-5-yl!methanone,

2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 2-2-(1-piperidinyl)ethoxy!pyridazin-5-yl!methanone, and

2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 3-2-(1-piperidinyl)ethoxy!pyridin-6-yl!methanone.

Particularly preferred embodiments of this invention are the compoundswhere: R₁ and R₂ are --OH, R₃ and R₄ are taken together to form apiperidine ring, X is 2,5 disubstituted pyridine, and Y is --CO--, asits hydrochloride salt, for example, 2-(4-hydroxyphenyl)-6-hydroxy-benzob!thien-3-yl! 2- 2-(1-piperidinyl)ethoxy!pyridin-5-yl!methanone.

The compounds of the current invention can be made according toprocedures well known in the art such as those detailed in U.S. Pat.Nos. 4,133,814; 4,418,068; and 4,380,635, the teachings of which areincorporated herein by reference.

In general, the process of preparing compounds of the present inventionstarts with a benzo b!thiophene having a 6-hydroxyl group and a2-(4-hydroxyphenyl) group. The starting compound is protected by methodsdetailed in T. Green, et al., "Protective Groups in Organic Synthesis,"Second Edition, John Wiley & Sons, New York, 1991. The protectedcompound is then acylated, and the resulting product subsequentlydeprotected to form the desired product of formula I, above. Examples ofthe preparation of such compounds are provided in the United StatesPatents listed above.

The formula I compounds which are carboxylic esters or sulfonates may beprepared by methods described in U.S. Pat. Nos. 5,393,763; 5,482,949 and5,482,949. Modifications to the above methods may be necessary toaccommodate reactive functionalities of particular substituents. Suchmodifications would be both apparent to, and readily ascertained by,those skilled in the art of organic chemistry.

As shown above, the nitrogen containing heterocycle groups defined bythe group "X" are oriented so that the bond at the top is intended to beread as attached to the ethoxy moiety and the bond at the bottom isintended to be read as bonded to the "Y" radical; that is: ##STR8##

Briefly, compounds of formula II ##STR9## where R₁ and R₂ have themeanings ascribed above, are acylated at the 3-position of thebenzothiophene nucleus with an activated carboxyl compounds of formulaIIIa and IIIb under standard Friedel-Crafts conditions. ##STR10## whereR³ and R⁴ are as defined above.

The compounds of formula II may be prepared in accordance with themethods described in U.S. Pat. No. 4,133,814. It is understood by thoseskilled in the art of organic chemistry that the ligands R₁ and R₂ mustbe compatible with the acylating conditions to form the compounds offormula I; thus a preferred intermediate of structure II would acompound where R₁ and R₂ are -OMe or other suitably protected hydroxylgroup.

The compounds of formula IIIa and IIIb are prepared from thecorresponding hydroxy acids by alkylating the hydroxy group with acompound of formula IV,

    Z--CH.sub.2 --CH.sub.2 --NR.sub.3 R.sub.4                  IV

in which Z is --Cl or --Br and R₃ and R₄ have their previous meanings,in the presence of a strong base, such as K₂ CO₃ or Cs₂ CO₃, followed byconversion by techniques well known in the art to the corresponding acidanhydride, acid chloride, or the like.

In general, the conditions for the acylation reaction between compoundsof type IIIa or IIIb and compounds of formula II involve the use of aLewis acid such as, AlCl₃, BF₃, and the like, in an appropriate solventsuch as a halogenated hydrocarbon, at temperatures ranging between aboutfrom 0° C. and 100° C.

Although shown above, for purposes of illustration, the activatedcarboxyl compounds IIIa and IIIb are acyl chlorides, the activatedcarboxyl compounds employed in the acylation reaction may also be mixedanhydrides, and the like, although acid chlorides are preferred.

In an alternate method for preparing the compounds of formula I,advantage is taken of the fact that halogen substituted, nitrogencontaining, aromatic heterocycles undergo facile substitution reactions.Chloro or bromo substituents next to a ring nitrogen can be easilydisplaced by alkyl hydroxy moieties in the presence of a strong base.Thus, a compound of formula V can be converted to a compound of formulaI by displacement of the chlorine with a compound of formula IV, where Zis hydroxyl. ##STR11##

The compounds of formula V are prepared by acylation of the benzob!thiophene (formula II) with an activated carboxylic acid moiety, e.g.,an acid chloride, of a chloro-substituted nitrogen heterocycliccarboxylic acid under Friedel-Crafts conditions described above. Anexample of this transformation is given below in Preparation 3.

The above chemical sequences for synthesis of the pyridine compounds areutilized for the preparation of other compounds of the present inventionwhich contain heterocyclic rings such as derived from pyridazine,pyrimidine, and pyrazine. The starting materials for the synthesis areknown in the art, e.g., the synthesis of 2-chloro-5-carboxy-pyridazinecan be found in "The Chemistry of Heterocyclic Compound, Pyridazines",Ed. Castle R. N., John Wiley & Sons, NYC, p. 432-433; the synthesis of2-chloro-5-cyano pyrimidine can be found in J. Org. Chem., 29, p. 1740;the synthesis of 2-carboxy-5-chloro pyrimidine can be found in Collect.Czech. Chem. Comm., 37, p. 1721, etc. It would be appearent to thoseskilled in the art of organic chemistry how these compounds could beulitized in the chemistry outlined above to obtain the compounds offormula I.

Other compounds of formula I where Y is a carbinol or methylene can beprepared by reduction of the carbonyl group in compounds obtained by theprocesses described above to the carbinol and further, if desired, tomethylene. Such reductions of the carbonyl group can be accomplishedstep-wise or from the carbonyl to methylene in a single step.

To obtain compounds of the present invention where Y is --CH(OH)--, thecarbonyl compound is reduced with LiAlH₄, NaBH₄, or the like inappropriate solvents such as chlorocarbons, tetrahydrofuran, ether, etc.at temperatures of ranging between about 0° C. and 30° C.

The carbinol group resulting from such reductions may be further reducedto the methylene by the action of silanes, e.g., triethylsilane, inappropriate solvents such as, methylene chloride or THF with a strongacid such as trifluoroacetic acid, at ambient temperatures.

Alternatively, the carbonyl compound may be reduced directly to themethylene by using LiAlH₄ in a high boiling solvent such aspropylbenzene at reflux temperatures.

The following illustrative Examples are provided to enable one skilledin the art to practice the present invention. However, these examplesare illustrative only, and should not be read as limiting the scope ofthe invention as it is defined by the appended claims.

Preparation 1 Preparation of 2-(4-Hydroxyphenyl)-6-hydroxybenzob!thien-3-yl! 2- 2-(1-piperidinyl)ethoxy!pyridin-5-yl!methanone

2-(4-Methoxyphenyl)-6-methoxybenzo b!thien-3-yl! 2-2-(1-piperidinyl)ethoxy!-pyridin-5-yl!methanone (1.8 g, 3.58 mmol) ofwas dissolved in 20 mL of dichloroethane and 3.1 mL (35.8 mmol) ofcondensed BCl₃ was added. The reaction mixture was stirred at ambienttemperature under a nitrogen atmosphere for forty-eight hours. Thereaction was quenched with 5 mL of MeOH and reaction was stirred for onehour. The reaction volume was evaporated to half the orginal volume andchromatographed on a silica gel column eluted with a linear gradientstarting with CHCl₃ and ending with CHCl₃ -MeOH (9:1) (v/v). The desiredfractions were determined by tlc, combined, and evaporated to dryness.The crude product was again chromatographed on a silica gel columneluted with CHCl₃ -MeOH (19:1). This yielded 540 mg of the titlecompound as a yellow-orange, amorphous solid.

Proton magnetic resonance spectrum: Consistent with the proposedstructure

MS: m/e=475 (M+) FD

Preparation 2 Preparation of 2-(4-Methoxyphenyl)-6-methoxybenzob!thien-3-yl! 2- 2-(1-piperidinyl)ethoxy!pyridin-5-yl!methanone

2-(4-Methoxyphenyl)-6-methoxybenzo b!thien-3-yl!2-chloropyridin-5-yl!methanone (1 g, 2.44 mmol) was dissolved in 10 mLof benzene and 950 mg (7.32 mmol) of 2-hydroxyethyl-1-piperidine wasadded. To the reaction mixture was added 85 mg (3.7 mmol) of sodium andthe reaction was stirred at ambient temperature under a nitrogenatmosphere until all the sodium dissolved. The reaction was heated to70° C. for two and half hours, then allowed to cool. To the reactionsolution was added 150 mL of EtOAc and the solution was washed threetimes with saturated aqueous Na₂ CO₃. The organic layer was separatedand evaporated to dryness. The crude product was chromatographed on asilica gel column eluted with a linear gradient beginning with CHCl₃ andending with CHCl₃ -MeOH (19:1). The desired fraction were determined bytlc, combined, and evaporated to dryness. This yielded the titlecompound as a yellow, amorphous solid.

Proton magnetic resonance spectrum: Consistent with the proposedstructure

Preparation 3 Preparation of 2-(4-Methoxyphenyl)-6-methoxybenzob!thien-3-yl! 2-chloropyridin-5-yl!methanone

2-(Methoxyphenyl)-6-methoxybenzo b!thiophene (7.8 g, 29 mmol) wasdissolved in 250 mL of dichloromethane. To this solution was added the2-chloro-5-carboxychloropyridine (from Preparation 4) in 20 mL ofdichloromethane. 31 g (232 mmol) of AlCl₃ was added to the reaction insix portions over a hour period. The reaction was allowed to proceed atambient temperature, under a nitrogen atmosphere for three and halfhours. The reaction was quenched by pouring it over ice and the organiclayer separated. The organic layer was washed three times with 500 mL ofsaturated, aqueous Na₂ CO₃, Dried by filtration through anhydrous Na₂SO₄ and evaporated to dryness. The crude product was chromatographed ona silica gel column eluted with EtOAc-hexane (1:9). The desiredfractions were determined by tlc, combined, and evaporated to dryness.This yielde the title compound as a yellow powder.

Proton magnetic resonance spectrum: Consistent with the

proposed structure

MS: m/e=409 (M+) FD

Elemental Analysis: Calc for C₂₂ H₁₆ ClNO₃ S: C, 64.47; H, 3.9; N, 3.42;Found: C, 64.77; H, 4.01; N, 3.16

Preparation 4 Preparation of 2-Chloro-5-carboxychloropyridine

2-Hydroxypyridine-5-carboxylic acid (4 g, 29 mmol) was dissolved 150 mLof dichloromethane, 50 mL of SOCl₂, and five drops of DMF. The ractionmixture was heated to reflux under a nitrogen atmosphere for sixteenhours. The reaction mixture was evaporated to an oily solid and usedwithout further purification or characterization (see Preparation 3).

Test Procedure General Preparation Procedure

In the examples illustrating the methods, a post-menopausal model wasused in which effects of different treatments upon circulating lipidswere determined.

Seventy-five day old female Sprague Dawley rats (weight range of 200 to225 g) are obtained from Charles River Laboratories (Portage, Mich.).The animals are either bilaterally ovariectomized (OVX) or exposed to aSham surgical procedure at Charles River Laboratories, and then shippedafter one week. Upon arrival, they are housed in metal hanging cages ingroups of 3 or 4 per cage and have ad libitum access to food (calciumcontent approximately 0.5%) and water for one week. Room temperature ismaintained at 22.2°±1.7° C. with a minimum relative humidity of 40%. Thephotoperiod in the room is 12 hours light and 12 hours dark.

Dosing Regimen Tissue Collection. After a one week acclimation period(therefore, two weeks post-OVX) daily dosing with test compound isinitiated. 17α-ethynyl estradiol or the test compound is given orally,unless otherwise stated, as a suspension in 1% carboxymethylcellulose ordissolved in 20% cyclodextrin. Animals were dosed daily for 4 days.Following the dosing regimen, animals are weighed and anesthetized witha ketamine: Xylazine (2:1, V:V) mixture and a blood sample is collectedby cardiac puncture. The animals are then sacrificed by asphyxiationwith CO₂, the uterus is removed through a midline incision, and a wetuterine weight is determined.

Cholesterol Analysis. Blood samples are allowed to clot at roomtemperature for 2 hours, and serum is obtained following centrifugationfor 10 minutes at 3000 rpm. Serum cholesterol is determined using aBoehringer Mannheim Diagnostics high performance cholesterol assay.Briefly the cholesterol is oxidized to cholest-4-en-3-one and hydrogenperoxide. The hydrogen peroxide is then reacted with phenol and4-aminophenazone in the presence of peroxidase to produce a p-quinoneimine dye, which is read spectrophotemetrically at 500 nm. Cholesterolconcentration is then calculated against a standard curve. The entireassay is automated using a Biomek Automated Workstation.

Uterine Eosinophil Peroxidase (EPO) Assay. Uteri are kept at 4° C. untiltime of enzymatic analysis. The uteri are then homogenized in 50 volumesof 50 mM Tris buffer (pH-8.0) containing 0.005% Triton X-100. Uponaddition of 0.01% hydrogen peroxide and 10 mMO-phenylenediamine (finalconcentrations) in Tris buffer, increase in absorbance is monitored forone minute at 450 nm. The presence of eosonophils in the uterus is anindication of estrogenic activity of a compound. The maximal velocity ofa 15 second interval is determined over the initial, linear portion ofthe reaction curve.

Source of Compound: 17α-ethynyl estradiol was obtained from SigmaChemical Co., St. Louis, Mo.

Hyperlipidemia:

Data presented in Table 1 show comparative results among ovariectomizedrats, rats treated with 17-a-ethynyl estradiol(EE₂), and rats treatedwith certain compounds of this invention. Although EE₂ caused a decreasein serum cholesterol when orally administered at 0.1 mg/kg/day, it alsoexerted a simulatory effect on the uterus so that EE₂ uterine weight wassubstantially greater than the uterine weight of the ovariectomizedanimals. This uterine response to an estrogen is well recognized in theart.

Not only did the compounds of the present invention reduce serumcholesterol compared to the ovariectomized animals, but the uterineweight was only minimally increased. Compared to estrogenic compoundsknown in the art, the benefit of serum cholesterol reduction withoutadversely affecting uterine weight is unusual and desirable.

As expressed in the data below, estrogenicity also was assessed byevaluating the response of eosinophil infiltration into the uterus. Thecompounds of this invention did not cause a large increase in the numberof eosinophils observed in the stromal layer of the ovariectomized, ratuteri. EE₂ caused a substantial and expected increase in eosinophilinfiltration.

The data presented in Table 1 reflect the response of five or six ratsper treatment group.

                  TABLE 1                                                         ______________________________________                                                          Increase in                                                                              Serum   Decrease in                                      Dose      Uterine Weight                                                                           Eosinophil                                                                            Cholesterol                              Compound                                                                              (mg/kg.sup.a)                                                                           (%)        (V.sub.max).sup.c                                                                     (%)                                      ______________________________________                                        EE2.sup.e                                                                             0.1       182.1*     211.1*  87.4*                                    Prep. 1 0.1       80.3*      48.0*   57.1*                                            1.0       91.4*      70.5*   66.1*                                            10.0      80.8*      55.8*   61.7*                                    ______________________________________                                         .sup.a 17a-Ethynyl estradiol                                                  .sup.b Uterine Weight % increase versus the ovarierectomized controls         .sup.c Eosinphil peroxidase Vmaxium                                           .sup.d Serum cholesterol decrease versus ovariectomized controls              *p < .05                                                                 

Osteoporosis Test Procedure

Following the General Preparation Procedure, infra, the rats are treateddaily for 35 days (6 rats per treatment group) and sacrificed by carbondioxide asphyxiation on the 36th day. The 35 day time period issufficient to allow maximal reduction in bone density, measured asdescribed herein. At the time of sacrifice, the uteri are removed,dissected free of extraneous tissue, and the fluid contents are expelledbefore determination of wet weight in order to confirm estrogendeficiency associated with complete ovariectomy. Uterine weight isroutinely reduced about 75% in response to ovariectomy. The uteri arethen placed in 10% neutral buffered formalin to allow for subsequenthistological analysis.

The right femurs are excised and digitilized x-rays generated andanalyzed by an image analysis program (NIH image) at the distalmetaphysis. The proximal aspect of the tibiae from these animals arealso scanned by quantitative computed tomography.

In accordance with the above procedures, compounds of the presentinvention and ethynyl estradiol (EE₂) in 20% hydroxypropylβ-cyclodextrin are orally administered to test animals.

In summary, ovariectomy of the test animals causes a significantreduction in femur density compared to intact, vehicle treated controls.Orally administered ethynyl estradiol (EE₂) prevented this loss, but therisk of uterine stimulation with this treatment is ever-present.

The compounds of the present invention prevent bone loss in a general,dose-dependent manner. Accordingly, the compounds of the presentinvention are useful for the treatment of post-menopausal syndrome,particularly osteoporosis.

MCF-7 Proliferation Assay

MCF-7 breast adenocarcinoma cells (ATCC HTB 22) are maintained in MEM(minimal essential medium, phenol red-free, Sigma, St. Louis, Mo.)supplemented with 10% fetal bovine serum (FBS) (V/V), L-glutamine (2mM), sodium pyruvate (1 mM), HEPES {(N- 2-hydroxyethyl!piperazine-N'-2-ethanesulfonic acid!10 mM}, non-essential amino acids and bovineinsulin (1 ug/mL) (maintenance medium). Ten days prior to assay, MCF-7cells are switched to maintenance medium supplemented with 10% dextrancoated charcoal stripped fetal bovine serum (DCC-FBS) assay medium) inplace of 10% FBS to deplete internal stores of steroids. MCF-7 cells areremoved from maintenance flasks using cell dissociation medium(Ca++/Mg++ free HBSS (phenol red-free) supplemented with 10 mM HEPES and2 mM EDTA). Cells are washed twice with assay medium and adjusted to80,000 cells/mL. Approximately 100 μL (8,000 cells) are added toflat-bottommicroculture wells (Costar 3596) and incubated at 37° C. in a5% CO₂ humidified incubator for 48 hours to allow for cell adherence andequilibration after transfer. Serial dilutions of drugs or DMSO as adiluent control are prepared in assay medium and 50 μL transferred totriplicate microcultures followed by 50 μL assay medium for a finalvolume of 200 μL. After an additional 48 hours at 37° C. in a 5% CO₂humidified incubator, microcultures are pulsed with tritiated thymidine(1 uCi/well) for 4 hours. Cultures are terminated by freezing at -70° C.for 24 hours followed by thawing and harvesting of microcultures using aSkatron Semiautomatic Cell Harvester. Samples are counted by liquidscintillation using a Wallac BetaPlace β counter. The compound inPreparation 1 is a potent inhibitor of the growth of MCF-7 cells with anIC₅₀ of 10 nM.

DMBA-Induced Mammary Tumor Inhibition

Estrogen-dependent mammary tumors are produced in female Sprague-Dawleyrats which are purchased from Harlan Industries, Indianapolis, Ind. Atabout 55 days of age, the rats receive a single oral feeding of 20 mg of7,12-dimethylbenz a!anthracene (DMBA). About 6 weeks after DMBAadministration, the mammary glands are palpated at weekly intervals forthe appearance of tumors. Whenever one or more tumors appear, thelongest and shortest diameters of each tumor are measured with a metriccaliper, the measurements are recorded, and that animal is selected forexperimentation. An attempt is made to uniformly distribute the varioussizes of tumors in the treated and control groups such thataverage-sized tumors are equivalently distributed between test groups.Control groups and test groups for each experiment contain 5 to 9animals.

Compounds of Formula I are administered either through intraperitonealinjections in 2% acacia, or orally. Orally administered compounds areeither dissolved or suspended in 0.2 mL corn oil. Each treatment,including acacia and corn oil control treatments, is administered oncedaily to each test animal. Following the initial tumor measurement andselection of test animals, tumors are measured each week by theabove-mentioned method. The treatment and measurements of animalscontinue for 3 to 5 weeks at which time the final areas of the tumorsare determined. For each compound and control treatment, the change inthe mean tumor area is determined.

Uterine Fibrosis Test Procedures

Test 1

Between 3 and 20 women having uterine fibrosis are administered acompound of the present invention. The amount of compound administeredis from 0.1 to 1000 mg/day, and the period of administration is 3months.

The women are observed during the period of administration, and up to 3months after discontinuance of administration, for effects on uterinefibrosis.

Test 2

The same procedure is used as in Test 1, except the period ofadministration is 6 months.

Test 3

The same procedure is used as in Test 1, except the period ofadministration is 1 year.

Test 4

A. Induction of fibroid tumors in guinea pig.

Prolonged estrogen stimulation is used to induce leiomyomata in sexuallymature female guinea pigs. Animals are dosed with estradiol 3-5 timesper week by injection for 2-4 months or until tumors arise. Treatmentsconsisting of a compound of the invention or vehicle is administereddaily for 3-16 weeks and then animals are sacrificed and the uteriharvested and analyzed for tumor regression.

B. Implantation of human uterine fibroid tissue in nude mice.

Tissue from human leiomyomas are implanted into the peritoneal cavityand or uterine myometrium of sexually mature, castrated, female, nudemice. Exogenous estrogen are supplied to induce growth of the explantedtissue. In some cases, the harvested tumor cells are cultured in vitroprior to implantation. Treatment consisting of a compound of the presentinvention or vehicle is supplied by gastric lavage on a daily basis for3-16 weeks and implants are removed and measured for growth orregression. At the time of sacrifice, the uteri is harvested to assessthe status of the organ.

Test 5

A. Tissue from human uterine fibroid tumors is harvested and maintained,in vitro, as primary nontransformed cultures. Surgical specimens arepushed through a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnRH, a compound of the present invention and vehicle.Levels of steroid hormone receptors are assessed weekly to determinewhether important cell characteristics are maintained in vitro. Tissuefrom 5-25 patients are utilized.

Activity in at least one of the above tests indicates the compounds ofthe present invention are of potential in the treatment of uterinefibrosis.

Endometriosis Test Procedure

In Tests 1 and 2, effects of 14-day and 21-day administration ofcompounds of the present invention on the growth of explantedendometrial tissue can be examined.

Test 1

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into three groups of equal numbers. The estrous cycleof all animals is monitored. On the day of proestrus, surgery isperformed on each female. Females in each group have the left uterinehorn removed, sectioned into small squares, and the squares are looselysutured at various sites adjacent to the mesenteric blood flow. Inaddition, females in Group 2 have the ovaries removed.

On the day following surgery, animals in Groups 1 and 2 receiveintraperitoneal injections of water for 14 days whereas animals in Group3 receive intraperitoneal injections of 1.0 mg of a compound of thepresent invention per kilogram of body weight for the same duration.Following 14 days of treatment, each female is sacrificed and theendometrial explants, adrenals, remaining uterus, and ovaries, whereapplicable, are removed and prepared for histological examination. Theovaries and adrenals are weighed.

Test 2

Twelve to thirty adult CD strain female rats are used as test animals.They are divided into two equal groups. The estrous cycle of all animalsis monitored. On the day of proestrus, surgery is performed on eachfemale. Females in each group have the left uterine horn removed,sectioned into small squares, and the squares are loosely sutured atvarious sites adjacent to the mesenteric blood flow.

Approximately 50 days following surgery, animals assigned to Group 1receive intraperitoneal injections of water for 21 days whereas animalsin Group 2 receive intraperitoneal injections of 1.0 mg of a compound ofthe present invention per kilogram of body weight for the same duration.Following 21 days of treatment, each female is sacrificed and theendometrial explants and adrenals are removed and weighed. The explantsare measured as an indication of growth. Estrous cycles are monitored.

Test 3

A. Surgical induction of endometriosis

Autographs of endometrial tissue are used to induce endometriosis inrats and/or rabbits. Female animals at reproductive maturity undergobilateral oophorectomy, and estrogen is supplied exogenously thusproviding a specific and constant level of hormone. Autologousendometrial tissue is implanted in the peritoneum of 5-150 animals andestrogen supplied to induce growth of the explanted tissue. Treatmentconsisting of a compound of the present invention is supplied by gastriclavage on a daily basis for 3-16 weeks, and implants are removed andmeasured for growth or regression. At the time of sacrifice, the intacthorn of the uterus is harvested to assess status of endometrium.

B. Implantation of human endometrial tissue in nude mice.

Tissue from human endometrial lesions is implanted into the peritoneumof sexually mature, castrated, female, nude mice. Exogenous estrogen issupplied to induce growth of the explanted tissue. In some cases, theharvested endometrial cells are cultured in vitro prior to implantation.Treatment consisting of a compound of the present invention supplied bygastric lavage on a daily basis for 3-16 weeks, and implants are removedand measured for growth or regression. At the time of sacrifice, theuteri is harvested to assess the status of the intact endometrium.

Test 4

A. Tissue from human endometrial lesions is harvested and maintained invitro as primary nontransformed cultures. Surgical specimens are pushedthrough a sterile mesh or sieve, or alternately teased apart fromsurrounding tissue to produce a single cell suspension. Cells aremaintained in media containing 10% serum and antibiotic. Rates of growthin the presence and absence of estrogen are determined. Cells areassayed for their ability to produce complement component C3 and theirresponse to growth factors and growth hormone. In vitro cultures areassessed for their proliferative response following treatment withprogestins, GnPa, a compound of the invention, and vehicle. Levels ofsteroid hormone receptors are assessed weekly to determine whetherimportant cell characteristics are maintained in vitro. Tissue from 5-25patients is utilized.

Activity in any of the above assays indicates that the compounds of thepresent invention are useful in the treatment of endometriosis.

Inhibition of Aortal Smooth Cell Proliferation/Restenosis

Test Procedure

Compounds of the present invention have capacity to inhibit aortalsmooth cell proliferation. This can be demonstrated by using culturedsmooth cells derived from rabbit aorta, proliferation being determinedby the measurement of DNA synthesis. Cells are obtained by explantmethod as described in Ross, J. of Cell Bio. 50: 172 (1971). Cells areplated in 96 well microtiter plates for five days. The cultures becomeconfluent and growth arrested. The cells are then transferred toDulbecco's Modified Eagle's Medium (DMEM) containing 0.5-2% plateletpoor plasma, 2 mML-glutamine, 100 U/ml penicillin, 100 mg mlstreptomycin, 1 mC/ml ³ H-thymidine, 20 ng/ml platelet-derived growthfactor, and varying concentrations of the present compounds. Stocksolution of the compounds is prepared in dimethyl sulphoxide and thendiluted to appropriate concentration (0.01-30 mM) in the above assaymedium. Cells are then incubated at 37° C. for 24 hours under 5% CO₂/95% air. At the end of 24 hours, the cells are fixed in methanol. ³ Hthymidine incorporation in DNA is then determined by scintillationcounting as described in Bonin, et al., Exp. Cell Res. 181:475-482(1989).

Inhibition of aortal smooth muscle cell proliferation by the compoundsof the present invention are further demonstrated by determining theireffects on exponentially growing cells. Smooth muscle cells from rabbitaortae are seeded in 12 well tissue culture plates in DMEM containing10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100mg/ml streptomycin. After 24 hours, the cells are attached and themedium is replaced with DMEM containing 10% serum, 2 mM L-glutamine, 100U/ml penicillin, 100 mg/ml streptomycin, and desired concentrations ofthe compounds. Cells are allowed to grow for four days. Cells aretreated with trypsin and the number of cells in each culture isdetermined by counting using a ZM-Coulter counter.

Activity in the above assays indicates that the compounds of the presentinvention are of potential in the treatment of restenosis.

The present invention also provides a method of alleviatingpost-menopausal syndrome in women which comprises the aforementionedmethod using compounds of Formula I and further comprises administeringto a woman an effective amount of estrogen or progestin. Thesetreatments are particularly useful for treating osteoporosis andlowering serum cholesterol because the patient will receive the benefitsof each pharmaceutical agent while the compounds of the presentinvention would inhibit undesirable side-effects of estrogen andprogestin. Activity of these combination treatments in any of thepost-menopausal tests, infra, indicates that the combination treatmentsare useful for alleviating the symptoms of post-menopausal symptoms inwomen.

Various forms of estrogen and progestin are commercially available.Estrogen-based agents include, for example, ethynyl estrogen (0.01-0.03mg/day), mestranol (0.05-0.15 mg/day), and conjugated estrogenichormones such as Premarin® (Wyeth-Ayerst; 0.3-2.5 mg/day).Progestin-based agents include, for example, medroxyprogesterone such asProvera® (Upjohn; 2.5-10 mg/day), norethylnodrel (1.0-10.0 mg/day), andnonethindrone (0.5-2.0 mg/day). A preferred estrogen-based compound isPremarin, and norethylnodrel and norethindrone are preferredprogestin-based agents.

The method of administration of each estrogen- and progestin-based agentis consistent with that which is known in the art. For the majority ofthe methods of the present invention, compounds of Formula I areadministered continuously, from 1 to 3 times daily. However, cyclicaltherapy may especially be useful in the treatment of endometriosis ormay be used acutely during painful attacks of the disease. In the caseof restenosis, therapy may be limited to short (1-6 months) intervalsfollowing medical procedures such as angioplasty.

As used herein, the term "effective amount" means an amount of compoundof formula I which is capable of alleviating the symptoms of the variouspathological conditions herein described. The specific dose of acompound administered according to this invention will, of course, bedetermined by the particular circumstances surrounding the caseincluding, for example, the compound administered, the route ofadministration, the state of being of the patient, and the pathologicalcondition being treated. Atypical daily dose will contain a nontoxicdosage level of from about 0.1 mg to about 1000 mg/day of a compound ofthe present invention, and more particularly will be from about 20 mg toabout 200 mg/day.

The compounds of this invention form pharmaceutically acceptable acidand base addition salts with a wide variety of organic and inorganicacids and bases and include the physiologically acceptable salts whichare often used in pharmaceutical chemistry. Such salts are also part ofthis invention. Typical inorganic acids used to form such salts includehydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,hypophosphoric and the like. Salts derived from organic acids, such asaliphatic mono and dicarboxylic acids, phenyl substituted alkanoicacids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, may also be used. Suchpharmaceutically acceptable salts thus include acetate, phenylacetate,trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate,phenylbutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate,caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate,glycollate, heptanoate, hippurate, lactate, malate, maleate,hydroxymaleate, malonate, mandelate, mesylate, nicotinate,isonicotinate, nitrate, oxalate, phthalate, teraphthalate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, propiolate, propionate, phenylpropionate, salicylate,sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite,bisulfite, sulfonate, benzene-sulfonate, p-bromophenylsulfonate,chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,p-toluenesulfonate, xylenesulfonate, tartarate, and the like. Apreferred salt is the hydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula I with an equimolar or excess amountof acid. The reactants are generally combined in a mutual solvent suchas diethyl ether or benzene. The salt normally precipitates out ofsolution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

Bases commonly used for formation of salts include ammonium hydroxideand alkali and alkaline earth metal hydroxides, carbonates, as well asaliphatic and primary, secondary and tertiary amines, aliphaticdiamines. Bases especially useful in the preparation of addition saltsinclude ammonium hydroxide, potassium carbonate, methylamine,diethylamine, ethylene diamine and cyclohexylamine.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions.

It is usually preferred to administer a compound of formula I in theform of an acid addition salt, as is customary in the administration ofpharmaceuticals bearing a basic group, such as the piperidino ring. Itis also advantageous to administer such a compound by the oral route.For such purposes the following oral dosage forms are available.

The compounds of this invention can be administered by a variety ofroutes including oral, rectal, transdermal, subucutaneus, intravenous,intramuscular, and intranasal. These compounds preferably are formulatedprior to administration, the selection of which will be decided by theattending physician. Thus, another aspect of the present invention is apharmaceutical composition comprising an effective amount of a compoundof Formula I, or a pharmaceutically acceptable salt thereof, optionallycontaining an effective amount of estrogen or progestin, and apharmaceutically acceptable carrier, diluent, or excipient.

The total active ingredients in such formulations comprises from 0.1% to99.9% by weight of the formulation. By "pharmaceutically acceptable" itis meant the carrier, diluent, excipients and salt must be compatiblewith the other ingredients of the formulation, and not deleterious tothe recipient thereof.

Pharmaceutical formulations of the present invention can be prepared byprocedures known in the art using well known and readily availableingredients. For example, the compounds of formula I, with or without anestrogen or progestin compound, can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivatives; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, andpolyvinyl-pyrrolidone; moisturizing agents such as glycerol;disintegrating agents such as calcium carbonate and sodium bicarbonate;agents for retarding dissolution such as paraffin; resorptionaccelerators such as quaternary ammonium compounds; surface activeagents such as cetyl alcohol, glycerol monostearate; adsorptive carrierssuch as kaolin and bentonite; and lubricants such as talc, calcium andmagnesium stearate, and solid polyethyl glycols.

The compounds also can be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for example, by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular physiological location,possibly over a period of time. The coatings, envelopes, and protectivematrices may be made, for example, from polymeric substances or waxes.

Compounds of formula I, alone or in combination with a pharmaceuticalagent of the present invention, generally will be administered in aconvenient formulation. The following formulation examples only areillustrative and are not intended to limit the scope of the presentinvention.

Formulations

In the formulations which follow, "active ingredient" means a compoundof formula I, or a salt or solvate thereof.

Formulation 1: Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________                                        Ingredient        Quantity (mg/capsule)                                       ______________________________________                                        Active ingredient 0.1-1000                                                    Starch, NF        0-650                                                       Starch flowable powder                                                                          0-650                                                       Silicone fluid 350 centistokes                                                                  0-15                                                        ______________________________________                                    

The formulation above may be changed in compliance with the reasonablevariations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 2: Tablets

    ______________________________________                                        Ingredient       Quantity (mg/tablet)                                         ______________________________________                                        Active ingredient                                                                                2.5-1000                                                   Cellulose, microcrystalline                                                                    200-650                                                      Silicon dioxide, fumed                                                                          10-650                                                      Stearate acid     5-15                                                        ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 2.5-1000 mg of active ingredientare made up as follows:

Formulation 3: Tablets

    ______________________________________                                        Ingredient         Quantity (mg/tablet)                                       ______________________________________                                        Active ingredient   25-1000                                                   Starch             45                                                         Cellulose, microcrystalline                                                                      35                                                         Polyvinylpyrrolidone                                                                             4                                                          (as 10% solution in water)                                                    Sodium carboxymethyl cellulose                                                                   4.5                                                        Magnesium stearate 0.5                                                        Talc               1                                                          ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 ml dose aremade as follows:

Formulation 4: Suspensions

    ______________________________________                                        Ingredient           Quantity (mg/5 ml)                                       ______________________________________                                        Active ingredient    0.1-1000  mg                                             Sodium carboxymethyl cellulose                                                                     50        mg                                             Syrup                1.25      mg                                             Benzoic acid solution                                                                              0.10      mL                                             Flavor                         q.v.                                           Color                          q.v.                                           Purified water to    5         mL                                             ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor, and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

An aerosol solution is prepared containing the following ingredients:

Formulation 5: Aerosol

    ______________________________________                                        Ingredient          Quantity (% by weight)                                    ______________________________________                                        Active ingredient    0.25                                                     Ethanol             25.75                                                     Propellant 22 Chlorodifluoromethane                                                               70.00                                                     ______________________________________                                    

The active ingredient is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to 30° C., and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remaining propellant. The valve units arethen fitted to the container.

Suppositories are prepared as follows:

Formulation 6: Suppositories

    ______________________________________                                        Ingredient       Quantity (mg/suppository)                                    ______________________________________                                        Active ingredient                                                                                250                                                        Saturated fatty acid glycerides                                                                2,000                                                        ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimal necessary heat. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows:

Formulation 7: Intravenous Solution

    ______________________________________                                        Ingredient            Quantity                                                ______________________________________                                        Active ingredient     50     mg                                               Isotonic saline       1,000  mL                                               ______________________________________                                    

The solution of the above ingredients is intravenously administered to apatient at a rate of about 1 mL per minute.

Formulation 8: Combination Capsule I

    ______________________________________                                        Ingredient    Quantity (mg/capsule)                                           ______________________________________                                        Active ingredient                                                                           50                                                              Premarin      1                                                               Avicel pH 101 50                                                              Starch 1500   117.50                                                          Silicon Oil   2                                                               Tween 80      0.50                                                            Cab-O-Sil     0.25                                                            ______________________________________                                    

Formulation 9: Combination Capsule II

    ______________________________________                                        Ingredient    Quantity (mg/capsule)                                           ______________________________________                                        Active ingredient                                                                           50                                                              Norethylnodrel                                                                              5                                                               Avicel pH 101 82.50                                                           Starch 1500   90                                                              Silicon Oil   2                                                               Tween 80      0.50                                                            ______________________________________                                    

Formulation 10: Combination Tablet

    ______________________________________                                        Ingredient     Quantity (mg/capsule)                                          ______________________________________                                        Active ingredient                                                                            50                                                             Premarin       1                                                              Corn Starch NF 50                                                             Povidone, K29-32                                                                             6                                                              Avicel pH 101  41.50                                                          Avicel pH 102  136.50                                                         Crospovidone XL10                                                                            2.50                                                           Magnesium Stearate                                                                           0.50                                                           Cab-O-Sil      0.50                                                           ______________________________________                                    

We claim:
 1. A compound of formula I ##STR12## or a pharmaceuticallyacceptable salt thereof wherein R¹ is selected from the group consistingofhydrogen, hydroxy, C₁ -C₄ alkoxy, --OC(O)O(C₁ -C₆ alkyl), --OC(O)(C₁-C₆ alkyl), and --OC(O)Ar wherein which Ar is selected from the groupconsisting ofunsubstituted phenyl, and phenyl substituted with --OSO₂(C₄ -C₆ straight chain alkyl); R² is selected from the group consistingofhydrogen, chloro, bromo, hydroxy, C₁ -C₄ alkoxy, --OC(O)O(C₁ -C₆alkyl), --OC(O)(C₁ -C₆ alkyl), and --OC(O)Ar wherein which Ar isselected from the group consisting ofunsubstituted phenyl, and phenylsubstituted with --OSO₂ (C₄ -C₆ straight chain alkyl ); R³ and R⁴ areindependently selected from C₁ -C₄ alkyl, or combine to form, togetherwith the nitrogen atom to which they are attached, ring selected fromthe group consisting ofpiperidino, pyrrolidino, and hexamethyleneimino;X is selected from the group consisting of ##STR13## Y is selected fromthe group consisting of --CO--,--CHOH--, and --CH₂ --.
 2. A compound asdefined by claim 1 selected from the group consisting of2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 2-2-(1-piperidinyl)ethoxy!pyrimidin-5-yl!methanone,2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 5-2-(1-piperidinyl)ethoxy!pyrimidin-2-yl!methanone,2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 2-2-(1-piperidinyl)ethoxy!pyrazin-5-yl!methanone,2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 2-2-(1-piperidinyl)ethoxy!pyridazin-5-yl!methanone, and2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl! 3-2-(1-piperidinyl)ethoxy!pyridin-6-yl!methanone; or a pharmaceuticallyacceptable salt thereof.
 3. A compound as defined by claim 1 having thename 2-(4-hydroxyphenyl)-6-hydroxybenzo b!thien-3-yl!- 2-2-(1-piperidinyl)ethoxy)pyridin-5-yl!methanone hydrochloride.
 4. Apharmaceutical composition comprising a compound according to claim 1,or a pharmaceutically acceptable salt thereof in combination with apharmaceutically acceptable carrier, diluent or excipient.
 5. Apharmaceutical composition as defined by claim 4 further comprising aneffective amount of estrogen.
 6. A pharmaceutical composition as definedby claim 4 further comprising an effective amount of progestin.
 7. Amethod for treating osteoporosis comprising administering to a woman inneed thereof an effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof.
 8. A method of treatingcardiovascular disease comprising administering to a woman in needthereof an effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof.
 9. A method according to claim8 wherein the cardiovascular disease is hyperlipidemia.
 10. A method forinhibiting estrogen-dependent cancer comprising administering to apatient in need thereof an effective amount of a compound of claim 1, ora pharmaceutically acceptable salt thereof.
 11. A method for inhibitingendometriosis comprising administering to a woman in need thereof aneffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof.
 12. A method for inhibiting aortal smoothmuscle cell proliferation comprising administering to a human in needthereof an effective amount of a compound of claim 1, or apharmaceutically acceptable salt thereof.
 13. A method for inhibitingrestenosis comprising administering to a human in need thereof aneffective amount of a compound of claim 1, or a pharmaceuticallyacceptable salt thereof.